The long-term goal of this project is to understand the viral and cellular determinants of HIV-1 particle assembly at the molecular level. We have recently demonstrated that the I or Interaction domain is an essential determinant of intracellular Gag protein complex formation and of plasma membrane localization of Gag. We propose a model in which I domain residues mediate Gag-RNA interaction and facilitate Gag-Gag multimerization. Intermediate complexes of Gag protein then are targeted to glycolipid-enriched subdomains of the plasma membrane (lipid rafts), where assembly takes place. To test this model, experiments will first define the role of the I domain in Gag-Gag multimerization The residues required for I domain function outside of the N-terminal subdomain of NC will be determined using site-directed mutagenesis, and their role in Gag-Gag multimerization defined using in vitro and cell-based assays. The protein content of dense versus light Gag protein particles will be analyzed, and the biochemical composition and morphology of light Gag particles (lacking the I domain) determined. Experiments in Specific Aim II will define the role of RNA in HIV-1 particle assembly. The RNA content of dense particles (containing and intact I domain) and of light particles (incorporating a disrupted I domain) will be quantified. The capacity of a defined panel of Gag protein constructs differing quantitatively in I domain function to bind to cellular RNA will next be analyzed. We will then define the role of the I domain in HIV-1 capsid formation using an RNA-dependent in vitro capsid assembly system. Specific Aim III will test the hypothesis that the interaction of Gag with lipid rafts requires Gag complex formation mediated by the I domain. Biochemical separation techniques and confocal microscopic methods will be utilized to define the influence of the I domain upon the Gag protein-lipid raft interaction. Pulse-chase analysis will be employed to characterize cytoplasmic Gag protein complexes that target to lipid rafts. The effect of the myristyl switch within MA to substitute for the I domain in lipid raft targeting will be tested. These experiments will answer fundamental questions relevant to HIV-1 particle assembly and may lead to unique opportunities for therapeutic intervention.